Factor VII fragment 82-128 and its use in blood-clotting disorders

ABSTRACT

The present invention relates to peptide of the amino acid sequence of formula (I): 
     
             82           85                  90                                   
 
    
         Glu Thr His Lys Asp Asp Gln Leu Ile                                   
                95                  100                                   
Cys Val Asn Glu Asn Gly Gly Cys Glu Gln                                   
                105                 110                                   
Tyr Cys Ser Asp His Thr Gly Thr Lys Arg                                   
                115                 120                                   
Ser Cys Arg Cys His Glu Gly Tyr Ser Leu                                   
                125         128                                           
Leu Ala Asp Gly Val Ser Cys Thr (SEQ ID NO. 1)                            
 
     and functional equivalents thereof in which amino acids in the above stated sequence are modified or absent and wherein at least one of the cysteine residues may be blocked or replaced and wherein any cysteine residues which remain unblocked or unreplaced may be present in a disulphide bonded state. The invention also includes slats and derivatives of said peptides. The peptides and their salts and derivatives are useful in the treatment of blood clotting disorders.

The present invention is concerned with peptide reagents andcompositions thereof which reduce blood clot formation.

Blood clotting relies upon a series or cascade of activating reactionsto produce the ultimate fibrin clot. The cascade leading to fibrinformation may be triggered initially in two different ways--by contactwith abnormal surfaces (the "intrinsic pathway") or by traumatization ofblood vessels which causes secretion of the lipoprotein known as "tissuefactor" or TF (the "extrinsic pathway"). The present invention isprimarily concerned with the extrinsic blood clotting pathway.

Coagulation factor VII(FVII) is a plasma glycoprotein of M_(r) =50,000consisting of a single polypeptide chain with 406 amino acid residues(F. S. Hagen et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 2412-2416; P.J. O'Hara et al., Proc. Natl. Acad. Sci. USA, 1987, 84: 5158-5162). Thezymogen form is converted to the fully enzymatically active formFVII_(a), by factor X and other coagulation proteases through hydrolysisof a single peptide bond Arg¹⁵² -Ile¹⁵³. This results in formation of anenzyme with two polypeptide chains which are held together by a singledisulphide bond. The light chain of 152 amino acid residues contains atits amino terminal part the γ-carboxy-glutamic acid (Gla) domain,followed by two epidermal growth factor-like domains (EGF-1 and EGF-2).The heavy chain consists of 254 residues and contains the serineprotease catalytic domain. The function of FVII_(a) is activation offactor X (FX) by complexation with tissue factor (TF) in the presence ofCa²⁺ on a phospholipid membrane surface. Activation of FX to FX_(a)leads to formation of blood clots by the extrinsic pathway.Additionally, the complex FVII_(a) /TF can activate factor IX to factorIX_(a) and lead to clotting through the intrinsic pathway.

Activation of the extrinsic pathway for blood clot formation is theprimary event leading to fibrin formation and is thus of primeimportance in the pathogenesis of arteriosclerotic lesions and inreocclusion and restenosis following endarterectomy. One way ofproviding potential therapeutic agents capable of preventing the primaryevent in blood clot formation through the extrinsic pathway would thusbe to identify peptides derived from the primary structure of FVII whichare capable of inhibiting the complex formation between FVII, TF and FX,which is necessary for FX activation.

Comparatively little is known about the mechanism and sites ofinteraction of FVII with TF and FX at the molecular level. Nevertheless,a number of FVII-derived peptides have been identified which possesssome inhibitory activity with respect to FX activation and which are notinhibitors of the enzymatic activity of FVII_(a). Peptides correspondingto sequence portions between the Gla and EGF-1 domains as well as fromthe catalytic domain, were disclosed in WO 9107432 (Board of Regents,The University of Texas System). A particular peptide from the EGF-2domain, as well as another peptide from the heavy chain were disclosedin WO 9003390 (Corvas, Inc.). Furthermore, we have disclosed in aco-pending application (PCT/GB94/01315) certain small peptides from theEGF-1 and mainly from the EGF-2 domains. In general the literaturesuggests that the Gla domain, EGF-1 domain and certain parts of theheavy chain are important in the recognition of FX and TF by FVII (P.Wildgoose et al., Proc. Natl. Acad. Sci. USA, 1990, 87: 7290-7294; W.Ruf et al., J. Biol. Chem., 1991, 266: 15719-15725; A. Kumar et al.,Eur. J. Biochem., 1993, 217: 509-518; S. Higashi et al., J. Biol. Chem.,1994, 269: 18891-19989).

We have investigated the EGF-2 domain of FVII and its role inprotein-protein interactions involving FVII. The arrangement of thethree disulphide bonds present in the EGF-2 domain of FVII is believedto follow a general pattern for proteins containing EGF-like domains andfor epidermal growth factor itself (H. Gregory, Nature, 1975, 257:325-327). For the FVII EGF-2 domain this corresponds to the followinghalf-cysteine pairs: Cys⁹¹ -Cys¹⁰², Cys⁹⁸ -Cys¹¹² and Cys¹¹⁴ -Cys¹²⁷. Wehave prepared various peptide analogues incorporating the EGF-2 domainof FVII by chemical synthesis and found that a fragment from Glu⁸² toThr¹²⁸ and variants of this have surprising activity in blocking theinteraction of FVII with TF in FX activation.

The fragment FVII 82-128 can be written in full as follows:

        82           85                  90                                           Glu Thr His Lys Asp Asp Gln Leu Ile                                                       95                  100                                       Cys Val Asn Glu Asn Gly Gly Cys Glu Gln                                                       105                 110                                       Tyr Cys Ser Asp His Thr Gly Thr Lys Arg                                                       115                 120                                       Ser Cys Arg Cys His Glu Gly Tyr Ser Leu                                                       125         128                                               Leu Ala Asp Gly Val Ser Cys Thr  SEQ ID NO. 1!                            

We have found that even when the three disulphide bonds of the nativestructure are absent in one of our synthetic EGF-2 domain peptides (byproviding 2-aminobutyric acid (Abu) replacements for the Cys residues toproduce a linear peptide) a potent inhibitor of FX activation isobtained.

The inhibition of FVII function by a number of synthetic peptidesincluding the above linear peptide was determined in a two-stagechromogenic assay as described (A. Kumar et al., 1991, J. Biol. Chem.,266@ 915-921). This consisted of incubation of peptide dilutions withmixtures of TF and FX, followed by addition of the test peptide anddetermination of the rate of FVII-induced FX activation by kineticmeasurement of FX amidolysis of a chromogenic substrate. Theconcentrations of peptides required to inhibit the rate of FX activationby 50% (IC₅₀) was determined, including appropriate controls (buffercontrol, non-related synthetic peptide) and the results are shown in thefollowing table.

    __________________________________________________________________________    Peptide position in factor VII IC.sub.50  (mM)                                __________________________________________________________________________    Asp.sup.48 -NH.sub.2           0.29                                           Asp.sup.104 -OH . .            0.62                                           Ser.sup.111 -NH.sub.2          >1                                             Ala.sup.206 -NH.sub.2          >1                                             Cys.sup.81 -OH!o- H-Cys.sup.72. . .                                                                          0.77                                           Cys.sup.102 -OH!- H-Cys.sup.91. . .                                                                          0.70                                           Cys.sup.127 -OH!- H-Cys.sup.114. . .                                                                         >1                                             Thr.sup.128 !,102,112,114,127 - Glu.sup.82 -. . .                                                            0.001                                                                          SEQ ID NO. 1!                                 __________________________________________________________________________

The surprisingly high inhibitory potency of the above linear EGF-2domain peptide, when compared to smaller peptides from the EGF-2 domainand other peptides previously proposed as useful inhibitors, suggestsdirectly for the first time that the EGF-2 domain is a functionallyimportant part of FVII/FVII_(a). A direct comparison of inhibitorypotency with other whole domains of FVII is difficult as little is knownabout such domains in the literature. At least for bovine FVII it wasfound (S. Higashi et al., J. Biol. Chem., 1994, 269: 18891-18898) that afragment corresponding to residues 1-85 of the light chain (presumablycomprising intact folded Gla and EGF-I domains) had an inhibitionconstant K_(i)(app) of 1 μM, whereas the K_(i)(app) of the intact lightchain was 0.35 μM with respect to inhibition of FVII_(a) amidolyticactivity. Furthermore, the same report claimed that a subfragment whichprobably contained the residues 86-137 (EGF-2 domain) was devoid ofinhibitory activity. We have measured a K_(i)(app) of approx. 6 μM forour linear 82-128 peptide, although a different assay system is used (FXactivation). Nevertheless, in both systems the K_(i)(app) values foractive site-inactivated FVII_(a) are approx. 10 nM. We have furtherestablished through kinetic means that the linear 82-128 peptide is notan active-site inhibitor of FVII but inhibits the TF/FVII/FX complexformation.

In WO-A-90/03390 referred to above, Corvas Inc. suggest that certainpeptides derived from the amino acid sequence of FVII (or FVIIa) mightbe useful in preventing the action of the fully formed FVIIa/TF complex.Two particular peptide sequences were disclosed in WO-A-90/03390 asbeing active in this respect. The sequence -Val Gly His Phe Gly Val- isbased upon amino acids nos. 372-377 of FVII which are situated near thecarboxy terminus. The other sequence is -Ser Asp His Thr Gly Thr Lys ArgSer Cys Arg- which is located at amino acids nos. 103-113 of FVII and ispart of the EGF-2 domain. Corvas Inc indicate that these peptides, andanalogues thereof, inhibit the cascade reaction initiated by theFVIIa/TF complex.

Furthermore, in Table 1, on page 14 of WO90/03390, it is indicated thatof the various regions of the EGF-2 domain, only -Ser Asp His Thr GlyThr Lys Arg Ser Cys-(103 to 112) was active and that the other regions,namely from amino acids 50 to 101 and 114 to 127, were totally inactivein inhibition of activation of Factor X by Factor VII and tissue factor.

However, we have shown that, contrary to the reported findings of CorvasInc., the region -Ser Asp His Thr Gly Thr Lys Arg Ser Cys- from 103 to112 is a rather poor inhibitor of the binding of Factor VII to tissuefactor.

Thus, in our co-pending International application PCT/GB94/01315referred to above we disclose peptide fragments from the region 91 to104 of the Factor VII sequence which are particularly active asinhibitors of the binding of Factor VII to tissue factor (TF) as well asuseful peptides from the regions 114-127. Peptides from the region 72-81are disclosed as acting synergistically with peptides from regions91-104 and 114-127. Several other fragments of the EGF-2 domain arereported as inactive.

As indicated above, we have now found surprisingly that peptidespossessing the FVII amino acid sequence 82-128 when in linearised form,that is lacking at least one of the three native Cys--Cys bridges, aremuch more active as inhibitors of FVII/TF binding than any of the FVIIfragments referred to above. This region includes as sub-regions severalsequences reported as inactive in the form of fragments. The fact thatlinearised EGF-2 peptides possess considerable inhibitory potency withrespect to FX activation compared to both linear and cyclicsub-fragments is particularly surprising. The linear EGF-2 peptides donot contain all the structural constraints imposed by the variousdisulphide bonds present in the native EGF-2 domain and can thus notnecessarily be expected to prefer the native conformation of the EGF-2domain in FVII.

According to the present invention we provide, as inhibitors of FVII/TFbinding, peptides of the amino acid sequence of formula (I)

        82           85                  90                                           Glu Thr His Lys Asp Asp Gln Leu Ile                                                       95                  100                                       Cys Val Asn Glu Asn Gly Gly Cys Glu Gln                                                       105                 110                                       Tyr Cys Ser Asp His Thr Gly Thr Lys Arg                                                       115                 120                                       Ser Cys Arg Cys His Glu Gly Tyr Ser Leu                                                       125         128                                               Leu Ala Asp Gly Val Ser Cys Thr  SEQ ID NO. 1!                            

and functional equivalents thereof in which amino acids in the abovestated sequence are modified or absent and wherein at least one of thecysteine residues may be blocked or replaced and wherein any cysteineresidues which remain unblocked or unreplaced may be present in adisulphide bonded state and salts and derivatives of said peptides.

The peptide should, in general, comprise the core region of amino acidresidues 91 to 112 and preferably includes additional terminal aminoacid sequences from the N and C terminal regions 82-90 and 113 to 128(as well as functional analogues, derivatives and salts of suchpeptides).

Typical N terminal additions to the above core region thus include Ile,Leu Ile, Gln Leu Ile, Asp Gln Leu Ile, Asp Asp Gln Leu Ile, Lys Asp AspGln Leu Ile, His Lys Asp Asp Gln Leu Ile, Thr His Lys Asp Asp Gln LeuIle, Glu Thr His Lys Asp Asp Gln Leu Ile. Typical C terminal additionsto the above core sequence include Arg, Arg Cys, Arg Cys His, Arg CysHis Glu, Arg Cys His Glu Gly, Arg Cys His Glu Gly Tyr, Arg Cys His GluGly Tyr Ser, Arg Cys His Glu Gly Tyr Ser Leu, Arg Cys His Glu Gly TyrSer Leu Leu, Arg Cys His Glu Gly Tyr Ser Leu Leu Ala, Arg Cys His GluGly Tyr Ser Leu Leu Ala Asp, Arg Cys His Glu Gly Tyr Ser Leu Leu Ala AspGly, Arg Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gly Val, Arg Cys HisGlu Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser, Arg Cys His Glu Gly Tyr SerLeu Leu Ala Asp Gly Val Ser Cys and Arg Cys His Glu Gly Tyr Ser Leu LeuAla Asp Gly Val Ser Cys Thr.

Particularly important modifications of the FVII 82-128 sequence arethose in which one or more of the cysteine residues are replaced, so asto eliminate disulphide bonding at that point. The SH groups of thecysteine residues may thus be blocked, eg. by alkylation by optionallysubstituted C₁₋₅ alkyl groups or C₆₋₁₂ aralkyl groups such astert.butyl, carboxymethyl, benzyl, acetamidomethyl, or pyridylethylgroups. Alternatively, the cysteine residue may be replaced by adifferent amino acid, preferably 2-aminobutyric acid (Abu), glycine,alanine or serine.

In general, if such modification is effected, preferably one, two orthree of the pairs of naturally linked cysteine residues are blocked orreplaced. The fully linearised derivatives in which all six cysteinesare blocked or replaced are especially preferred, notably that with Abuin place of each cysteine.

A number of conservative amino acid substitutions are recognised in theart as having little or no functional effect, notably Ser for Thr, Ilefor Leu and Lys for Arg. Such modifications are also included.

The terminal amino group may be `capped`, that is blocked by an inertgroup such as an acyl group, eg. a C₁₋₅ alkanoyl group, for exampleacetyl. The terminal carboxyl group may also be capped, for example byformation of carbamoyl group, eg. CONH₂, or an ester group, eg. a C₁₋₅alkoxycarbonyl group.

Salts of the peptides include physiologically acceptable acid additionsalts such as the hydrochloride.

The present invention also provides pharmaceutical compositionscontaining one or more of the peptides of the invention or saltsthereof.

The compositions according to the invention may be presented, forexample, in a form suitable for oral, nasal, parenteral or rectaladministration.

As used herein, the term "pharmaceutical" includes veterinaryapplications of the invention.

The compounds according to the invention may be presented in theconventional pharmacological forms of administration, such as tablets,coated tablets, nasal sprays, solutions, emulsions, powders, capsules orsustained release forms. Conventional pharmaceutical excipients as wellas the usual methods of production may be employed for the preparationof these forms. Tablets may be produced, for example, by mixing theactive ingredient or ingredients with known excipients, such as forexample with diluents, such as calcium carbonate, calcium phosphate orlactose, disintegrants such as corn starch or alginic acid, binders suchas starch or gelatin, lubricants such as magnesium stearate or talcum,and/or agents for obtaining sustained release, such ascarboxypolymethylene, carboxymethyl cellulose, cellulose acetatephthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets maybe produced by coating cores, obtained in a similar manner to thetablets, with agents commonly used for tablet coatings, for example,polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxideor sugar. In order to obtain sustained release or to avoidincompatibilities, the core may consist of several layers too. Thetablet-coat may also consist of several layers in order to obtainsustained release, in which case the excipients mentioned above fortablets may be used.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Nasal sprays may be formulated similarly in aqueous solution and packedinto spray containers either with an aerosol propellant or provided withmeans for manual compression. Capsules containing one or several activeingredients may be produced, for example, by mixing the activeingredients with inert carriers, such as lactose or sorbitol, andfilling the mixture into gelatin capsules.

Suitable suppositories may, for example, be produced by mixing theactive ingredient or active ingredient combinations with theconventional carriers envisaged for this purpose, such as natural fatsor polyethyleneglycol or derivatives thereof.

Dosage units containing the compounds of this invention preferablycontain 0.1-10 mg, for example 1-5 mg of the peptide of formula (I) orsalt thereof.

As indicated above, one aspect of the invention provides peptides(including the analogues or salts thereof) according to the inventionfor use in the treatment or prevention of blood clotting disorders orproblems. Blood clotting disorders include thrombosis (particularlyvascular thrombosis or deep vein thrombosis), acute myocardialinfarction, restenosis, reclosure, angina, cerebrovascular disease,peripheral arterial occlusive disease, hypercoagulability and pulmonaryembolism. The peptides according to the invention can also be used toprevent occurrence of blood clotting problems caused by, for example,injury to blood vessels during thrombolytic therapy, grafting surgery,vessel patency restoration etc. Blood clotting disorders may betriggered by sepsis due to production of TNF-α or IL-1.

In a still further aspect, the present invention also provides a methodof treatment of blood disorders in the mammalian, preferably human,animal body, said method comprising administering to said body one ormore peptides of formula (I) or analogues or salts thereof. Prophylacticmethods of treatment are also provided, whereby a peptide according tothe invention is administered to a patient to prevent or reduce theoccurrence of possible blood clotting problems, for example duringsurgery or other invasive techniques. The peptide will of coursenormally be administered in the form of a pharmaceutically acceptablecomposition.

In another aspect, the present invention provides a process for thepreparation of peptides which comprise the sequence of formula (I) orare derived therefrom, or analogues or salts thereof.

The peptides of the present invention can be prepared by methods knownin the art. Typically, the desired sequences are assembled bysolid-phase peptide synthesis. Standard procedures for the synthesisstrategy employed for the examples of this invention are described in E.Atherton & R. C. Sheppard, Solid phase peptide synthesis: a practicalapproach, 1989, IRL Press, Oxford. For example, a synthesis resinderivatised with an acid-labile linker group is first esterified with aprotected form of the C-terminal amino acid. The amino-protecting groupis then removed and the second amino acid in the sequence is coupledusing a suitable condensation reagent. Amino acids with semi-permanentamino protecting groups and permanent protecting groups for functionalside chains are employed. Amino-deprotection and coupling cycles arethen repeated in alternating steps until the sequence of interest isassembled. Finally the permanent side-chain protecting groups areremoved and the peptide is cleaved from the synthesis resin usuallysimultaneously through treatment with a suitable acidic reagent.Alternatively, the peptides can be synthesised through solution peptidesynthesis, either in a step-wise manner from the carboxyl terminusand/or through the application of segment condensation methods, eitheremploying comprehensive (see e.g. Y. Nishiuchi et al., (1992) in`Peptides: Chemistry and Biology`, J. A. Smith and J. E. Rivier (eds.),pp. 911-913, ESCOM, Leiden) or minimal protection/ ligation (see e.g. P.E. Dawson et al., Science, 1994, 266: 776-779) strategies. Combinedsolution--solid phase segment condensation approaches can also beapplied (see e.g. H. Benz, Synthesis, 1994, 337-358).

Generally the reactive groups present (for example amino, thiol and/orcarboxyl) will be protected during overall synthesis as indicated above.The final step in the synthesis will thus be the deprotection of aprotected derivative of the peptide of the invention.

A wide choice of protecting groups for amino- acids are known and areexemplified in Schroder, E., and Lubke, K., The Peptides, Vols. 1 and 2,Academic Press, New York and London, 1965 and 1966; Pettit, G. R.,Synthetic Peptides, Vols. 1-4, Van Nostrand, Reinhold, N.Y. 1970, 1971,1975 and 1976; Houben-Weyl, Methoden der Organischen Chemie, Synthesevon Peptiden, Band 15, Georg Thieme Verlag, Stuttgart 1974; Amino Acids,Peptides and Proteins, Vol.4-8, The Chemical Society, London 1972, 1974,1975 and 1976; Peptides, Synthesis-physical data 1-6, Wolfgang Voelter,Eric Schmidt-Siegman, Georg Thieme Verlage Stuttgart, NY, 1983; ThePeptides, Analysis, synthesis, biology 1-7, Ed: Erhard Gross, JohannesMeienhofer, Academic Press, NY, San Fransisco, London; Solid phasepeptide synthesis 2nd ed., John M. Stewart, Janis D. Young, PierceChemical Company.

Thus, for example amine protecting groups which may be employed includeprotecting groups such as carbobenzoxy (hereinafter also designated Z)t-butoxycarbonyl (hereinafter also designated Boc) and9-fluorenylmethoxycarbonyl (hereinafter also designated Fmoc). It willbe appreciated that when the peptide is built up from the C-terminalend, an amine-protecting group will be present on the α-amino group ofeach new residue added and will need to be removed selectively prior tothe next coupling step. One particularly useful group for such temporaryamine protection is the Fmoc group which can be removed selectively bytreatment with piperidine in an organic solvent.

Carboxyl protecting groups which may, for example be employed includereadily cleaved ester groups such as benzyl (Bz1), p-nitrobenzyl (ONb),or t-butyl (OtBu) groups as well as the linkers on solid supports, forexample p-alkoxybenzylalcohol groups linked to polystyrene.

Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt),acetamidomethyl (Acm), t-butylsulphenyl (SBu^(t)) and 2-pyridylsulphenyl(Npys).

It will be appreciated that a wide range of other such groups exists as,for example, detailed in the above-mentioned literature references, andthe use of all such groups in the hereinbefore described processes fallwithin the scope of the present invention.

A wide range of procedures exists for removing amine- andcarboxyl-protecting groups. These must, however, be consistent with thesynthetic strategy employed. The side chain protecting groups must bestable to the conditions used to remove the temporary α-amino protectinggroup prior to the next coupling step.

Amine protecting groups such as Boc and carboxyl protecting groups suchas tBu may be removed simultaneously by acid treatment, for example withtrifluoro acetic acid. In general, where the pairs of cysteine residuesin native FVII 82-128 remain in the SH form, they are preferably causedto interact oxidatively to form a disulphide bond.

There are two common ways of forming intramolecular disulphide bonds insynthetic peptides. In the single-step approach the reduced linearpeptide is submitted directly to oxidation with an oxidant such as airor dimethylsulphoxide (J. P. Tam et al., J. Am. Chem. Soc., 1991, 113:6657-6662). Alternatively, oxidation via disulphide exchange withreduced/oxidised glutathione or similar reagents can be employed (K. O.Johanson et al., J. Biol. Chem., 1981, 256: 445-450). The directapproach depends on the ability of the peptide in question to fold intothe native state and often results in by-products with undesireddisulphide pairings. The other approach is sequential in nature andinvolves regioselective formation of individual disulphide bonds (K.Akaji et al., Tetrahedron Lett., 1992, 33: 1073-1076). This necessitatesthe use of different thiol protecting groups for prospective pairs ofCys residues. The protecting groups are chosen to permit step-wiseselective deprotection in the presence of pre-formed disulphide bonds.Such an approach is preferred for peptides containing multipledisulphide bonds described in this invention and has also been appliedto the chemical synthesis of EGF-2 peptides from coagulation factor IX(Y. Yang et al., Protein Science, 1994, 3: 1267-1275).

The FVII EGF-2 peptides of this invention may also be produced throughgenetic engineering. In the first step synthetic oligonucleotidesincluding the sequences encoding the polypeptides in question(specifically nucleotide sequences within position 459-599 according toF. S. Hagen et al., Proc. Natl. Acad. Sci. USA, 1986, 83: 2412-2416,FIG. 2) may be produced by standard methods. Alternatively, cDNA orgenomic DNA digest libraries may be produced (e.g. as described in P. J.O'Hara et al., Proc. Natl. Acad. Sci. USA, 1987, 84: 5158-5162) andscreened with a labelled probe specific for DNA sequences encoding theEGF-2 domain. In the second step, DNA thus produced is then cloned intosuitable expression vectors, which are used to transform microorganisms(typically Escherichia coli bacteria or certain yeasts). Finally thesemicroorganisms are cultured and the polypeptide products from theinserted foreign DNA are isolated from the cultures through proteinchemistry methods well known in the art.

Thus, according to a further feature of the invention we provide arecombinant DNA molecule comprising an expression vector containing DNAaccording to the sequence:

    GAG ACG CAC AAG GAT GAC CAG CTG ATC                                                                    SEQ ID No. 6!                                        TGT GTG AAC GAG AAC GGC GGC TGT GAG                                           CAG TAC TGC AGT GAC CAC ACG GGC ACC                                           AAG CGC TCC TGT CGG TGC CAC GAG GGG                                           TAC TCT CTG CTG GCA GAC GGG GTG TCC                                           TGC ACA                                                                   

and modifications thereof coding for peptides according to theinvention.

The following Examples are given by way of illustration only:

EXAMPLE 1

Abu⁹¹,98,102,112,114,127 - FVII Glu⁸² -Thr¹²⁸ ! SEQ ID No. 2!Thepeptidyl resin corresponding to the above sequence was assembled onFmoc-Thr(Bu^(t))- TentaGel ACS resin! (0.1 mmol;2-methoxy-4-alkoxybenzyl alcohol handle; from Rapp Polymere GmbH,Tubingen, Germany) using an Applied Biosystems model 433A peptidesynthesiser. Fmoc-deprotection was achieved with conductivity monitoringusing 20% piperidine in N,N-dimethylformamide (DMF). The washing solventwas DMF. The first 20 residues (from carboxyl terminus) were coupledwith 10-fold molar excess of Fmoc-amino acids amd2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate/1-hydroxybenzotriazole(HOBt)/Pri^(i) ₂ NEt in N-methylpyrrolidone using 75 min couplingcycles. The remaining 20 residues were assembled using double couplings(75 min each cycle). Prior to Fmoc-deprotection at each sequenceposition capping was carried out using a solution of acetic anhydride(4.7% v/v) /Pr^(i) ₂ NEt (2.2% v/v)/HOBt (0.2% w/v) in DMF. The aminoacid side-chain protecting groups used were:2,2,5,7,8-pentamethyl-chroman-6-sulphonyl for Arg, trityl for Asn, Glnand His, t-butyloxycarbonyl for Lys, t-butyl for Asp, Glu, Ser, Thr andTyr. The final Fmoc-deprotected and washed (Et₂ O) peptidyl resin wasdried in vacuo. An aliquot (40 mg) was treated with a mixture containingphenol, 1,2-ethanedithiol, thioanisole, water and CF₃ COOH(0.75:0.25:0.5:0.5:10, w/v/v/v/v) for 4 h. Resin residue was thenfiltered off and washed with small quantities of neat CF₃ COOH. Thecombined filtrate and washings were concentrated in vacuo. The crudepeptide was then obtained by trituration with Et₂ O and collection ofthe precipitate by centrifugation and further washes with Et₂ O. Thedried crude product (13 mg) was fractionated by semi-preparative RP-HPLC(Vydac C₁₈ column, 1×25 cm; 3 mL/min, 15 to 30% MeCN in 0.1% aq CF₃ COOHover 2 h). Appropriate peak fractions were pooled and lyophilised toafford 2.0 mg of pure peptide. Analytical RP-HPLC: t_(R) =29.4 min,purity 96% (Vydac 2l8TP54, 0.46×25 cm, 1 mL/min, 15-30% MeCN in 0.1% aqCF₃ COOH over 40 min, λ=215 nm). ES-MS: M!=5056.1±0.35, C₂₁₃ H₃₃₇ N₆₅O₇₈ =5056.42. N-Terminal sequence analysis (ABI gas-phase sequencer)over the first 6 residues confirmed the presence of the desired peptide.Amino acid analysis: Asx 5.94(6), Thr 3.98(4), Ser 4.09(4), Glx 5.84(6),Gly 5.03(5), Ala 1.03(1), Val 1.99 (2), Ile 0.96(1), Leu 3.03(3), Tyr2.02(2), His 2.97(3), Lys 2.00(2), Arg 2.02(2), Abu 6.10 (6).

EXAMPLE 2

Disulphide-cyclo Cys⁹¹ -Cys¹⁰² !-Abu⁹⁸,112,114,127 - FVII Glu⁸² -Thr¹²⁸! SEQ ID No. 3!

The peptidyl resin corresponding to the sequence Abu⁹⁸,112,114,127 -FVII Glu⁸² -Thr¹²⁸ ! was assembled in a similar fashion to thecorresponding peptidyl resin of Example 1 with the exception that Cys⁹¹and Cys¹⁰² were introduced as the Fmoc-Cys(SBu^(t))-OH derivatives. Thecomplete peptidyl resin was worked up and an aliquot (80 mg dry weight)treated with the same acidolysis reagent as in Example 1. Afterprecipitation from Et₂ O, the product was dissolved in 0.1% aq CF₃ COOHwith the aid of ultrasonication and was then lyophilised to yield 16.2mg white solid material. This was redissolved in 0.1% aq CF₃ COOH (4mL), filtered and applied to an RP-HPLC column (Vydac 218TP1022, 2.2×25cm). The column was developed at 5 mL/min with a gradient of 20 to 35%MeCN in 0.1% aq CF₃ COOH over 2 h. Appropriate peak fractions werepooled and lyophilised to afford pure Cys⁹¹,102 (SBu^(t))-protectedmaterial. Analytical RP-HPLC: t_(R) =24.5 min, purity 97% (Vydac218TP54, 0.46×25 cm, 1 mL/min, 20-35% MeCN in 0.1 aq CF₃ COOH over 40min, λ=215 nm).

This material was dissolved in a mixture of 2,2,2-trifluoroethanol andwater (95:5, v/v; 2 mL) and tri-n-butylphosphine (2 drops) was added.The mixture was shaken for 1 h and was then evaporated in vacuo. Theresidue was precipitated from Et₂ O, collected by centrifugation andfurther washed with Et₂ O in a similar fashion. The dried reducedmaterial was dissolved in a mixture of CF₃ COOH and dimethylsulphoxide(95:5, v/v, 2 mL) and oxidation was allowed to proceed for 90 min. CF₃COOH was then removed from the mixture by rotary evaporation underwater-pump vacuum. The residual solution was diluted with an equalvolume of water and was applied directly to an RP-HPLC column (Vydac218TP1022, 2.2×25 cm). The column was developed at 5 mL/min with agradient of 20 to 30% MeCN in 0.1% aq CF₃ COOH over 2 h. Appropriatepeak fractions corresponding to the disulphide Cys⁹¹,102 oxidisedmaterial were pooled and lyophilised to provide the title compound.Analytical RP-HPLC: t_(R) =13.3 min, purity 95% (Vydac 218TP54, 0.46×25cm, 1 mL/min, 20-35% MeCN in 0.1% aq CF₃ COOH over 40 min, λ=215 nm).ES-MS: M!=5089.5±0.62, C₂₁₁ H₃₃₁ N₆₅ O₇₈ S₂ =5090.44. Amino acidanalysis: Asx 5.80(6), Thr 3.85(4), Ser 4.40(4), Glx 5.70 (6), Gly5.11(5), Cys 1.79(2), Ala 1.00(1), Val 2.06(2), Ile 0.85 (1), Leu 3.04(3), Tyr 1.96 (2), His 2.92(3), Lys 1.87(2), Arg 2.01 (2), Abu 4.63(4).

EXAMPLE 3

Disulphide-cyclo Cys¹¹⁴ -Cys¹²⁷ !-Abu⁹¹,98,102,112, - FVII Glu⁸² -Thr¹²⁸! SEQ ID No. 4!

The peptidyl resin corresponding to the sequence Abu⁹¹,98,102,112, -FVII Glu⁸² -Thr¹²⁸ ! was assembled in a similar fashion to thecorresponding peptidyl resin of Example 1 with the exception that Cys¹¹⁴and Cys¹²⁷ were introduced as the Fmoc-Cys(SBu^(t))-OH derivatives. Thecomplete peptidyl resin was worked up and treated with the sameacidolysis reagent as in Example 1. After precipitation from Et₂ O, theproduct was dissolved in 0.1% aq CF₃ COOH and was then lyophilised. Thismaterial was used directiy in the next step. Analytical RP-HPLC: t_(R)=28.3 min, major peak (Vydac 218TP54, 0.46×25 cm, 1 mL/min, 20-35% MeCNin 0.1% CF₃ COOH over 40 min, λ=215 nm).

The Cys¹¹⁴,127 (SBu^(t))-protected material was resuspended in H₂ O.Concentrated aq ammonia solution (2 drops) was then added to bring thepeptide material into solution. Pilot experiments using this proceduredemonstrated that base treatment lead to very rapid removal of thet-butylsulphenyl protecting groups, as well as formation of the desireddisulphide bond. The soluuon was then filtered and applied to an RP-HPLCcolumn (Vydac 218TP1022, 2.2×25 cm). The column was developed at 5mL.min with a gradient of 20 to 35% MeCN in 0.1% aq CF₃ COOH over 2hours. Appropriate peak fractions were pooled and lyophilised to affordpure title compound. Analytical RP-HPLC: t_(R) =30.3 min, purity 92%(Vydac 218TP54, 0.46×25 cm, 1 mL/min, 15-30% MeCN in 0.1% aq CF₃ COOHover 40 min, λ=215 nm). ES-MS: M!=5090.0, C₂₁₁ H₃₃₁ N₆₅ O₇₈ S₂ =5090.44.Amino acid analysis: Asx 5.66(6), Thr 4.02(4), Ser 4.47(4), Glx 5.72(6),Gly 5.31(5), Cys 2.28(2), Ala 1.11(1), Val 1.76(2), Ile 0.97(1), Leu3.20(3), Tyr 1.90(2), His 2.98(3), Lys 1.96(2), Arg 1.85(2), Abu3.81(4).

EXAMPLE 4

Disulphide-cyclo Cys⁹⁸ -Cys¹¹² !-Abu⁹¹,102,114,127 - FVII Glu⁸² -Thr¹²⁸! SEQ ID No. 5!

The peptidyl resin corresponding to the sequence Abu⁹¹,102,114,127 -FVII Glu⁸² -Thr¹²⁸ ! was assembled in a similar fashion to thecorresponding peptidyl resin of Example 1 with the exception that Cys⁹⁸and Cys¹¹² were introduced as the Fmoc-Cys(Acm)-OH derivatives. Thecomplete peptidyl resin was worked up and an aliquot treated with thesame acidolysis reagent as in Example 1. After precipitation from Et₂ O,the product was dissolved in 0.1% aq CF₃ COOH and was then lyophilised.The residue was redissolved in O.1% aq CF₃ COOH, filtered and applied toan RP-HPLC column (Vydac 2187TP1022, 2.2×25 cm). The column wasdeveloped at 5 mL/min with a gradient of 15 to 30% MeCN in 0.1% aq CF₃COOH over 2 hours. Appropriate peak fractions were pooled andlyophilised to afford pure Cys⁹⁸,112 (Acm)-protected material. MALDI-TOFMS: M+H!⁺ =5235.1, C₂₁₇ H₃₄₃ N₆₇ O₈₀ S₂ =5234.65.

Acm-deprotection was carried out as follows: The Cys⁹⁸,112(Acm)-protected material (2 mg) was dissolved in 30% aq AcOH (0.4 mL)and Hg(OAc)₂ (3 mg) was added. The solution was agitated at ambienttemperature for 4 hours. 2-Mercaptoethanol (10 μL) was then added andthe mixture was left overnight. It was then diluted with 0.1% aq CF₃COOH and the grey precipitate containing mercury salts was removed bycentrifugation and decantation. MALDI-TOF MS analysis of the supernatantindicated the presence of the peptide as a mercury adduct. More2-mercaptoethanol (10 μL) was therefore added and the mixture was leftto stand overnight. The mixture was then clarified as before, desaltedby preparative RP-HPLC and lyophilised. The crude reduced (ascertainedusing the quantitative Ellman's test, refer G. L. Elmman (1959) Arch.Biochem. Biophys. 82, 70) material was used directly in the next step.

Oxidation was carried out as follows: The Acm-deprotected reducedmaterial was dissolved in an aqueous buffered solution (0.1 M Trisacetate, pH 7.8; 5 mL) containing a mixture of oxidised (0.5 mM) andreduced (0.25 mM) glutathione. The reaction was followed by analyticalRP-HPLC, which indicated rapid conversion. After 4 hours, the solutionwas lyophilised. The residue was redissolved in 0.1% aq CF₃ COOH andapplied to an RP-HPLC column (Vydac 218TP1022, 2.2×25 cm). The columnwas developed at 5 mL/min with a gradient of 15 to 30% MeCN in 0.1% aqover 40 min). Appropriate peak fractions were pooled and lyophilised toafford pure title compound (0.30 mg). Analytical RP-HPLC: t_(R) =27.8min (Vydac 218TP54, 0.46×25 cm, 1 mL/min, 15-30% MeCN in 0.1% aq CF₃COOH over 40 min, λ=215 nm). MALDI-TOF MS: M+H!⁺ =5089.9, C₂₁₁ H₃₃₁ N₆₅O₇₈ S₂ =5090.44. Amino acid analysis: Asx 5.24(6), Thr 3.81(4), Ser4.64(4), Glx 5.34(6), Gly 5.66(5), Cys 1.91(2), Ala 1.49(1), Val2.33(2), Ile 0.71(1), Leu 3.56(3), Tyr 2.14(2), His 2.64(3), Lys1.55(2), Arg 1.84(2), Abu 4.07(4).

EXAMPLE 5

Disulphide-cyclo Cys⁹¹ -Cys¹⁰²,Cys⁹⁸ -Cys¹¹²,Cys¹¹⁴ -Cys¹²⁷ !- FVIIGlu⁸² -Thr¹²⁸ ! SEQ ID No. 1!

Cys⁹⁸,112 (Acm) - FVII Glu⁸² -Thr¹²⁸ !: The peptidyl resin correspondingto the sequence FVII Glu⁸² -Thr¹²⁸ ! was assembled in a similar fashionto the corresponding peptidyl resin of Example 1 with the exception thatCys⁹¹, Cys¹⁰², Cys¹¹⁴ and Cys¹²⁷ were introduced as the Fmoc-Cys(Trt)-OHderivatives, whereas Cys⁹⁸ and Cys¹¹² were introduced as theFmoc-Cys(Acm)-OH derivatives. The final peptidyl resin was cleaved anddeprotected using the same procedures as in Example 1(4 hours reactiontime). After Et₂ O precipitation and drying, the crude material wasredissolved in 0.1% aq CF₃ COOH and was lyophilised. The residue waschromatographed by RP-HPLC (Vydac 218TP1022, 2.2×25 cm) at 5 mL/minusing gradient elution from 15 to 30% MeCN in 0.1% aq CF₃ COOH over 2hours. Appropriate peak fractions were pooled and lyophilised to affordpure title compound. Analytical RP-HPLC: t_(R) =28.6 min (Vydac 218TP54,0.46×25 cm, 1 mL/min, 15-30% MeCN in 0.1% aq CF₃ COOH over 40 min).ES-MS: M!=5306.4, C₂₁₃ H₃₃₅ N₆₇ O₈₀ S₆ =5306.81.

Disulphide-cyclo Cys91-Cys¹⁰², Cys¹¹⁴ -Cys¹²⁷ !-Cys⁹⁸,112 (Acm)- FVIIGlu⁸² -Thr¹²⁸ !:Cys⁹⁸,112 (Acm)- FVII Glu⁸² - Thr¹²⁸ ! was oxidised at4° C. with a peptide concentration of 0.1 mg/mL in a buffered aqueoussolution (0.1 M Tris acetate, pH 7.8) containing a mixture of oxidised(0.50 mM) and reduced (0.25 mM) glutathione. The reaction was followedby analytical RP-HPLC, which indicated essentially complete conversionafter 2 hours to the title compound (t_(R) 26.3 min using the samecondtions as for the precursor). The mixture was then lyophilised,redissolved in 0.1% aq CF₃ COOH (3 mL) and chromatographed by RP-HPLCanalogously to the reduced precursor, with the exception that thegradient was developed over 40 min at a flow rate of 9 mL/min. Peakfractions containing pure title compound were pooled and lyophilised.

Disulphide-cyclo Cys⁹¹ -Cys¹⁰², Cys⁹⁸ -Cys¹¹², Cys¹¹⁴ -Cys¹²⁷ !- FVIIGlu⁸² -Thrl¹²⁸ !: disulphide-cyclo Cys⁹¹ -Cys¹⁰², Cys¹¹⁴ -Cys¹²⁷!-Cys⁹⁸,112 (Acm)- FVII Glu⁸² -Thr¹²⁸ ! was dissolved in H₂ O (250 μL).MeOH (250 μL), anisole (2 μL), and AcOH (30 μL) were then added. To thissolution cyanogen iodide (5 mg) was added and the reaction mixture wasagitated overnight in the dark. RP-HPLC and MALDI-TOF MS analysis of themixture indicated essentially complete conversion. RP-HPLC: t_(R) =13.8min (starting material at 12.8 min) (Vydac 218TP54, 0.46×25 cm, 1mL/min, 20-35% MeCN in 0.1% aq CF₃ COOH over 40 min). The reaction wasthen quenched by addition of excess 0.1 M aq Na₂ S₂ O₃ and was dilutedto 2 mL with O.1% aq CF₃ COOH. This solution was purified by preparativeRP-HPLC using the same conditions as for the precursor. Appropriate peakfractions containing the pure title compound were pooled andlyophilised. Analytical HP-HPLC t_(R) =27.2 min, purity 95% (Vydac218TP54, 0.46×25 cm, 1 mL/mim, 15-30% MeCN in 0.1% aq CF₃ COOH over 40min, μ=215 nm) . MALI-TOF MS: M+H!⁺ 5159.6, C₂₀₇ H₃₁₉ N₆₅ O₇₈ S₆=5158.66. Amino acid analysis: Asx 5.81(6), Thr 3.85(4), Ser 4.63(4),Glx 6.00(6), Gly 5.33(5), Cys 5.66(6), Ala 1.06(1), Val 2.15(2), Ile1.03(1), Leu 2.93(3), Tyr 1.95(2), His 2.48(3), Lys 2.11(2), Arg2.01(2).

EXAMPLE 6

Inhibition of factor VIIa/tissue factor-catalysed factor X activation bycompounds of Examples 1 to 5

This was determined using a two-stage chromogenic biochemical in vitroassay, essentially as described (A. Kumar et al., 1991, J. Biol. Chem,266, 915). In short: peptides were pre-incubated with lipidated TF (5pM; from American Diagnostica, Inc., Greenwich, Conn., USA) and calcium(5 mM) for 10 min prior to addition of FVIIa (5 pM; from Novo NordiskA/S, Gentofte, Denmark) and FX (20 nm: from Enzyme Research Laboratory,South Bend, Ind., USA). Reactions were terminated by addition of EDTA(50 mM) and formation of FXa was monitored using the chromogenic FXasubstrate S2765 (0.4 mM; from Chromogenix, Moindal, Sweden) andmeasuring the absorbance increase at 405 nm in a microtitre platereader. The concentration for half-maximal inhibition (IC50) wasdetermined from dose-inhibition curves.

    ______________________________________                                                           IC.sub.50  (μM)                                         ______________________________________                                        Example 1   (fully linear)                                                                             1.3 ± 0.7 (n = 16).sup.a                          Example 2   (Cys.sup.91 -Cys.sup.102)                                                                  0.26                                                 Example 3   (Cys.sup.114 -Cys.sup.127)                                                                 0.29                                                 Example 4   (Cys.sup.98 -Cys.sup.112)                                                                  0.14                                                 Example 5   (fully cyclic)                                                                             0.08                                                 ______________________________________                                         .sup.a Value corrected for net peptide content. IC.sub.50  value based on     peptide weight was 3 μM.                                              

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Thr His Lys Asp Asp Gln Leu Ile Cys Va - #l Asn Glu Asn Gly             #                15                                                           - Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Th - #r Lys Arg Ser Cys             #            30                                                               - Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gl - #y Val Ser Cys Thr             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:10                                                               (D) OTHER INFORMATION:/pro - #duct= "10"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:17                                                               (D) OTHER INFORMATION:/pro - #duct= "17"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:21                                                               (D) OTHER INFORMATION:/pro - #duct= "21"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:31                                                               (D) OTHER INFORMATION:/pro - #duct= "31"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:33                                                               (D) OTHER INFORMATION:/pro - #duct= "33"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:46                                                               (D) OTHER INFORMATION:/pro - #duct= "46"                            #"Xaa represents Abu"                                                         #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Thr His Lys Asp Asp Gln Leu Ile Xaa Va - #l Asn Glu Asn Gly             #                15                                                           - Xaa Glu Gln Tyr Xaa Ser Asp His Thr Gly Th - #r Lys Arg Ser Xaa             #            30                                                               - Xaa His Glu Gly Tyr Ser Leu Leu Ala Asp Gl - #y Val Ser Xaa Thr             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:17                                                               (D) OTHER INFORMATION:/pro - #duct= "17"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:31                                                               (D) OTHER INFORMATION:/pro - #duct= "31"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:33                                                               (D) OTHER INFORMATION:/pro - #duct= "33"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:46                                                               (D) OTHER INFORMATION:/pro - #duct= "46"                            #"Xaa represents Abu"                                                         #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Thr His Lys Asp Asp Gln Leu Ile Cys Va - #l Asn Glu Asn Gly             #                15                                                           - Xaa Glu Gln Tyr Cys Ser Asp His Thr Gly Th - #r Lys Arg Ser Xaa             #            30                                                               - Xaa His Glu Gly Tyr Ser Leu Leu Ala Asp Gl - #y Val Ser Xaa Thr             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:10                                                               (D) OTHER INFORMATION:/pro - #duct= "10"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:17                                                               (D) OTHER INFORMATION:/pro - #duct= "17"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:21                                                               (D) OTHER INFORMATION:/pro - #duct= "21"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:31                                                               (D) OTHER INFORMATION:/pro - #duct= "31"                            #"Xaa represents Abu"                                                         #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Thr His Lys Asp Asp Gln Leu Ile Xaa Va - #l Asn Glu Asn Gly             #                15                                                           - Xaa Glu Gln Tyr Xaa Ser Asp His Thr Gly Th - #r Lys Arg Ser Xaa             #            30                                                               - Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gl - #y Val Ser Cys Thr             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 47 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:10                                                               (D) OTHER INFORMATION:/pro - #duct= "10"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:21                                                               (D) OTHER INFORMATION:/pro - #duct= "21"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:33                                                               (D) OTHER INFORMATION:/pro - #duct= "33"                            #"Xaa represents Abu"                                                         -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION:46                                                               (D) OTHER INFORMATION:/pro - #duct= "46"                            #"Xaa represents Abu"                                                         #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Glu Thr His Lys Asp Asp Gln Leu Ile Xaa Va - #l Asn Glu Asn Gly             #                15                                                           - Cys Glu Gln Tyr Xaa Ser Asp His Thr Gly Th - #r Lys Arg Ser Cys             #            30                                                               - Xaa His Glu Gly Tyr Ser Leu Leu Ala Asp Gl - #y Val Ser Xaa Thr             #        45                                                                   - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 141 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                            (A) DESCRIPTION:   / - #desc = "recombinant DNA"                    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GAGACGCACA AGGATGACCA GCTGATCTGT GTGAACGAGA ACGGCGGCTG TG - #AGCAGTAC         60                                                                          - TGCAGTGACC ACACGGGCAC CAAGCGCTCC TGTCGGTGCC ACGAGGGGTA CT - #CTCTGCTG        120                                                                          #                 141AC A                                                     __________________________________________________________________________

We claim:
 1. A peptide of the amino acid sequence of formula I

        82           85                  90                                           Glu Thr His Lys Asp Asp Gln Leu Ile                                                       95                  100                                       Cys Val Asn Glu Asn Gly Gly Cys Glu Gln                                                       105                 110                                       Tyr Cys Ser Asp His Thr Gly Thr Lys Arg                                                       115                 120                                       Ser Cys Arg Cys His Glu Gly Tyr Ser Leu                                                       125         128                                               Leu Ala Asp Gly Val Ser Cys Thr (SEQ ID NO. 1)                            

of human factor VII and functional equivalents thereof in which aminoacids in the region 82-90 and 113-182 are modified or absent; or theamino acids Ser, Thr, Ile, Leu, Lys, and Arg are each optionallymodified by the substitutions Ser for The, Ile for Leu, and Lys for Argand vice versa; or both, and optionally wherein at least one cysteineresidue in the sequence of formula (I) is blocked or replaced andwherein cysteines which remain unblocked or unreplaced are present in adisulfide bonded state; or a salt or derivative of said peptide.
 2. Apeptide as claimed in claim 1 wherein one or more cysteine residues arereplaced or blocked.
 3. A peptide as claimed in claim 2 wherein the thiogroup of said cysteine residue is blocked by an optionally substitutedC₁₋₅ alkyl or C₆₋₁₂ aralkyl group.
 4. A peptide as claimed in claim 2wherein said cysteine residue is replaced by glycine, alanine, serine or2-aminobutyric acid.
 5. A peptide as claimed in claim 2 wherein all sixcysteine residues are replaced or blocked.
 6. A peptide as claimed inclaim 5 wherein the cysteine residues are replaced by 2-aminobutyricacid.
 7. A peptide as claimed in claim 1 wherein the terminal aminogroup or the terminal carboxyl group or both terminal amino group andterminal carboxyl group are capped by an inert group.
 8. A peptide asclaimed in claim 7 wherein the terminal amino capping group is a C₁₋₅alkanoyl group and wherein the terminal carboxyl capping group is acarbamoyl or ester group.
 9. A peptide as claimed in claim 1 wherein anycysteine residue remaining unblocked or unreplaced is present in anatural half-cystine disulphide bonded state.
 10. A pharmaceuticalcomposition containing a peptide as claimed in claim 1 together with apharmaceutically acceptable carrier.
 11. A pharmaceutical composition asclaimed in claim 10 in a form suitable for oral, nasal, parenteral orrectal administration.
 12. A peptide as claimed in claim 1 forinhibition of blood coagulation.
 13. A method of inhibiting bloodcoagulation in the mammalian body said method comprising administeringto said body a peptide as claimed in claim
 1. 14. A recombiant DNAmolecule comprising an expression vector containing DNA according to thesequence:

    GAG ACG CAC AAG GAT GAC CAG CTG ATC TGT GTG AAC GAG AAC                                                         (SEQ ID NO 6)                               GGC GGC TGT GAG CAG TAC TGC AGT GAC CAC ACG GGC ACC AAG                       CGC TCC TGT CGG TGC CAC GAG GGG TAC TCT CTG CTG GCA GAC                       GGG GTG TCC TGC ACA                                                       

and modifications thereof coding for the peptide as claimed in claim 1.